To help determine the biological basis of learning and memory, our research seeks to understand how plasticity in the cerebellum mediates eyeblink conditioning. To pursue this goal, we have recorded in vitro learning-specific synaptic and membrane changes in Purkinje cells of the rabbit cerebellar cortex following eyelid conditioning. There are two limitations to this approach. First, a number of different cerebellar lobules are involved in eyelid conditioning. Second, it is not possible to confirm whether cells in which changes are detected were involved in the behavior. There is now a potential solution to these problems. First, all cerebellar lobules project to deep cerebellar nuclei and there is growing evidence that changes in neural activity, synapse number and gene expression occur within the anterior interpositus nucleus as a result of eyeblink conditioning. Second, there are retrograde neuronal track tracing techniques able to identify deep cerebellar neurons involved in the eyeblink. However, although these cells can be visualized, there is a dense perineuronal net surrounding adult deep cerebellar nuclei that prevents recording from these cells. To overcome all of these limitations, we are developing a rat pup cerebellar slice preparation in which we can visualize and record from eyeblink-related neurons in the anterior interpositus following injection of a fluorescent retrograde tracer into the eyelid. With this preparation in hand, we will address two specific aims: (1) identify and characterize anterior interpositus neurons involved in the rat eyeblink;and (2) determine the changes in synaptic and intrinsic membrane properties of identified anterior interpositus neurons that occur as a result of eyeblink conditioning. This work is important because of the impact it will have on understanding the synaptic and intrinsic membrane changes underlying learning and memory in a well-understood behavioral model. The proposed work is innovative because it develops a slice preparation that for the first time provides data on the role of identified neurons involved in learning and memory. It combines a novel approach - visually identifiable response-related neurons in the anterior interpositus nucleus with advanced in vitro electrophysiological recording techniques in a well-understood model of learning and memory - rat eyeblink conditioning. PUBLIC HEALTH RELEVANCE: The evidence is clear that damage to the deep cerebellar nuclei in animals and humans has a major impact on both motor and non-motor forms of learning and memory. Consequently, examining changes in the synaptic and intrinsic membrane properties of cells in the cerebellar nuclei as a function of eyeblink conditioning in rats may provide insights into the biological mechanisms of learning and memory. By identifying these changes, the proposed work has the potential to help understand the pathogenesis of failures in learning and memory and to develop intervention strategies for those failures.